The mucosal surfaces (i.e., gut, mouth) of vertebrates are continuously exposed to endogenous and exogenous microbes. Throughout evolutionary time, specific immunoglobulin (Ig) isotypes have become specialized to protect such mucosal sites. In mammals and birds, IgA predominates at mucosal surfaces, whereas IgG and IgM provide systemic protection. Although mucosal Igs were thought to evolve in four legged (tetrapod) species, during the previous funding period, we discovered that fish contain an Ig isotype, IgT, that is specialized for gut mucosal immunity. Thereafter we discovered extraordinarily similar IgT responses occurred in other mucosal sites, including the skin and gills. Based on these results, we hypothesize that IgT responses in all fish mucosal areas (gut, gills and skin) operate under the guidance of primordially conserved principles. Thus, the overarching goal of this renewal is to test the hypothesis that pathogen-specific IgT responses are locally induced (Aim 1) in a T cell-dependent fashion (Aim 2), and lead to the generation of effector IgT+ B cells that generate protective IgT responses and maintain tissue homeostasis through the production of IL-10 (Aim 3). In addition, as we have found bacterial microbiota from gill, gut and skin prevalently coated by IgT, we hypothesize that commensal microbiota induce local T-cell independent IgT responses (Aim 2) and, that this IgT homeostatically regulates the microbiome at mucosal sites (Aim 3). Delineating strategies of immunoglobulin-based mucosal immunity in our model will not only illuminate unresolved paradigms of mucosal immune responses in non-tetrapods, but will also provide insights into responses. More specifically, our proposed studies are expected to: a) provide novel insights into the extrafollicular pathways by which IgA production is regulated in response to pathogens and microbiota; b) contribute towards the understanding of the roles of CD4+ T cells in generating IgA responses to pathogens and microbiota; c) provide ground-breaking knowledge on the mechanisms by which mucosal Igs control microbiota and its composition (microbiome) at multiple mucosal sites. Thus, the aims of this renewal are: AIM 1. To investigate the inductive and effector sites involved in the generation of IgT responses and, to analyze the existence of a common mucosal immune system (CMIS) in fish. To uncover the IgT inductive and effector sites, in vivo pathogen-induced B and T cell proliferative responses along with the visualization of B-Tcell interactions will be assessed in all MALTs and the spleen, using normal fish and a novel splenectomized fish model. Here we will also assess the existence of a common mucosal immune system (CMIS) by determining whether B/T cell responses induced at one mucosal site lead to effector responses at other distant mucosal areas. AIM 2. To investigate the T-cell dependency of IgT responses to pathogens and microbiota. Using an innovative CD4-T cell depletion fish model, we will assess fish mortality rate, pathogen load, B cell proliferative and pathogen-specific Ig responses. We will also evaluate the effect of CD4-T cell depletion on the composition of all gut, gill and skin microbiomes and the percentage of bacterial microbiota coated with IgT and IgM. To gain insight into the mechanisms by which IgT responses are modulated in the absence of CD4+ T cells, we will also investigate the effect of CD4+ T cell depletion on the IgT sequence repertoire of all mucosal sites of na?ve and infected fish. AIM 3. To investigate the requirement of IgT+ B cells for the control of pathogens and bacterial microbiota. The goals of this aim will be accomplished using a powerful and novel IgT+ B cell depletion fish model. Fish mortality rates, tissue damage, pathogen load, complement activation and pathogen-specific systemic and mucosal Ig responses will be assessed in IgT+ B cell depleted fish upon pathogen challenge. Using the same fish models we will also evaluate changes in the microbiome composition of all mucosal sites and the percentage of bacterial microbiota coated with IgT and IgM.